Sequential modulations of distractor-related interference  have been taken to reflect conflict-induced attentional focusing. To dismiss an alternative interpretation based on integration and retrieval of low-level features, it is important to exert experimental control of stimulus and response feature sequences. This has been achieved by considering only trials associated with complete feature changes. Furthermore, distractors from two different perceptual dimensions, such as stimulus location and shape, have been combined in the same experiment to investigate the question of specificity vs. generality of conflict adaptation. With this method feature sequence control can be exerted, in principle, without disregarding data from feature repetition trials. However, such control may be insufficient when the distractor dimensions overlap semantically. In two experiments we found evidence consistent with the assumption that semantic generalization of stimulus features, such as between a stimulus presented at a left-sided location and a stimulus shape pointing to the left, may yield a between-dimension Gratton effect. These findings raise doubts about inferring generalized attentional conflict adaptation when semantically related distractor dimensions are used. This congruency effect has been found to be reduced after incongruent as compared to after congruent predecessor trials, a sequential modulation often referred to as the Gratton effect. The Gratton effect has been observed in a variety of different tasks, such as the Stroop task  of the event file, and mismatches between co-activated representations and current perceptual input or response demands are assumed to interfere with response selection. Therefore, feature integration theory predicts performance impairments on partial repetition trials  compared to complete feature repetition or alternation trials.An alternative view to this attentional adaptation account was put forward by Hommel et al. , see MayThe possible role of stimulus and response feature sequences for the Gratton effect becomes apparent if one looks at the sequences of congruency levels in standard interference tasks. Consider a typical Simon task, in which participants perform a binary classification of a non-spatial target feature, such as judging whether a given stimulus is black or white, by pressing one of two response keys. On each trial, the stimulus is presented in one of two possible locations, each of which spatially corresponds to the location of one of the responses, a left one and a right one, say. A given trial is congruent if stimulus and response locations fall on the same side and incongruent if stimulus and response locations fall on opposite sides. As can be seen in Table Previous research has tried to deconfound the sequence of congruency levels and the sequences of stimulus and response features. One approach is characterized by using a larger number of stimuli and responses and confining the sequential congruency analysis to trials in which all discriminative stimulus and response features differ from the preceding trial. Applying this approach bears the risk of associating congruent and incongruent stimuli with different degrees of distractor-target or distractor-response contingencies, thereby making it possible to account for a Gratton effect in terms of contingency level switch costs rather than conflict adaptation  speaks to the question of specificity vs. generality of conflict adaptation mechanisms congruency and to (mis)match between stimulus and response location as location-(in)congruency. In addition to within-dimension Gratton effects , Kunde and Wühr observed—albeit smaller—reductions of location- and direction-based congruency effects after incongruent trials regarding the other distractor dimension.In some studies, however, between-dimension Gratton effects were successfully obtained  middle quarters of Table abstract (feature sequence) match trials and the latter trials as abstract (feature sequence) mismatch trials.To gain evidence about semantic generalization of stimulus features that belong to different distractor dimensions it is instructive to look at the relationship of congruency level sequences and abstract feature sequences in more detail. Inspection of Table It is important to note that whereas on abstract match trials the sequence of the response either matches or mismatches both abstract feature sequences , on abstract mismatch trials the sequence of the response matches one of the abstract feature sequences and mismatches the other one. Thus, on abstract match trials, a conjoined repetition or alternation of the response and the abstract left/right feature regarding one between-dimension transition  is always associated with a conjoined repetition or alternation of the response and the abstract left/right feature regarding the reversed transition . And a partial repetition regarding the response and the abstract left/right feature regarding one between-dimension transition is always associated with a partial repetition regarding the response and the abstract left/right feature regarding the reversed between-dimension transition. In contrast, on abstract mismatch trials, whenever there is a conjoined repetition or alternation of the response and the abstract left/right feature regarding one between-dimension transition, there is a partial repetition regarding the response and the abstract left/right feature regarding the reversed between-dimension transition.As a consequence of this contingency, on abstract match trials both semantic sequence effects should work in the same direction, whereas on abstract mismatch trials they should work in opposite directions. Assuming that semantic location-direction transitions and direction-location transitions yield effects of comparable strength—that is, a stimulus location integrated in an event file is activated by perceiving a corresponding arrow direction to roughly the same amount as an arrow direction integrated in an event file is activated by perceiving a corresponding stimulus location—semantic sequence effects should add up to zero on abstract feature sequence mismatch trials. On the corollary assumption of comparable effect strength, semantic generalization should therefore yield a between-dimension Gratton effect selectively on abstract match trials and an absence thereof on abstract mismatch trials. The semantic generalization hypothesis thus predicts between-dimension Gratton effects on abstract match trials and, assuming comparable efficacy of semantic generalization for both dimension transition directions, the absence of a between-dimension Gratton effect on abstract mismatch trials.In the first experiment of the current study, we explored the role of semantic generalization for between-dimension Gratton effects by combining stimulus location and pointing direction of the stimulus shape, each of which could vary between the values left and right, as distractor dimensions, thereby producing the contingencies displayed in Table Two female and 13 male students of the Helmut-Schmidt-University/University of the Federal Armed Forces Hamburg participated in exchange for partial course requirements. They ranged in age from 19 to 28 years. The experiments of the current study were conducted in accordance with the ethical guidelines of the German Psychological Society (Deutsche Gesellschaft für Psychologie) and the Declaration of Helsinki of the World Medical Association. Formal ethics approvals for the described kind of research are not required by the guidelines of the German Psychological Association or the World Medical Association.The stimuli were presented on a 17-in. monitor with a refresh rate of 60 Hz. A dark gray background was used. Stimuli were arrows pointing either to the left or to the right, which extended 3.0 cm horizontally and 2.5 cm vertically. As mentioned above, we inserted blocks of trials, in which participants responded to arrow direction, amongst the experimental blocks, in which stimulus color had to be judged. We refer to the former blocks as intermediate blocks, and to the latter blocks as critical blocks. In the critical blocks, arrows were presented in either blue or yellow color and occurred either 2.4 cm to the left or to the right of the screen center (nearest edge) on the horizontal midline. In the intermediate blocks the arrows were white stimuli and occurred in the screen center. Two response keys were used. They were located on an external keyboard and extended 1.0 × 1.0 cm. One key was located 4.0 cm to the left and the other 4.0 cm to the right of the keyboard's saggital midline, which was placed perpendicular to the screen and aligned with the screen center. The left response key was pressed with the index or middle finger of the left hand; the right response key was pressed with the index or middle finger of the right hand. During an experimental block the fingers remained on the keys. Regarding the critical blocks, odd-numbered participants were instructed to press the left key for yellow and the right key for blue. This assignment was reversed for even-numbered participants. In the intermediate blocks, participants were to respond to the direction of the arrow with the spatially corresponding key press. Figure Participants sat approximately 50 cm from the computer screen. Each trial began with the presentation of a fixation cross in the center of the screen (0.3 × 0.3 cm). After a period of 500 ms, an arrow stimulus was presented. In the critical blocks, the color (blue vs. yellow), the location (left vs. right), and the direction (left vs. right) of the arrow were chosen randomly on each trial. In the intermediate blocks, arrow direction was again chosen randomly on each trial. Participants were instructed to classify the stimulus by pressing the assigned response key as quickly as possible while avoiding errors. Immediately after a response key was pressed, the stimulus and the fixation cross disappeared from the screen. In case of a correct response the next trial started 500 ms after the response with the presentation of the fixation cross. In case of an incorrect response, the German word “falsch” (“incorrect”) occurred for 800 ms slightly below the screen center. Then the trial was repeated with an identical stimulus. Repetitions of incorrect trials were not counted as trials .At the beginning of the experiment participants received written instructions. After a practice block of 30 trials, which was structurally identical to the critical blocks, participants were presented with 24 critical blocks of 35 trials each. In advance of each critical block, an intermediate block of 16 trials was administered. Only critical blocks were subjected to the statistical analysis. Between blocks, participants were allowed to rest for some time. A complete session took between 40 and 45 min.The first three trials of each critical block were considered “warm-up” trials and not analyzed. Furthermore, we excluded data from the first two trials following an error as well as RTs associated with an incorrect response or smaller than 200 ms or larger than 1200 ms. 0.2% of the data were eliminated by excluding RT outliers.N) and the preceding (N − 1) trial. To investigate the semantic generalization hypothesis, we analyzed between-dimension sequential congruency effects depending on match vs. mismatch of the abstract feature sequences.Two sets of analysis, an overall analysis of within-dimension and between-dimension Gratton effects, and a semantic feature sequence analysis, were conducted. In the overall analysis, trials were classified depending on location-congruency and direction-congruency on the current  with repeated measures on the factors direction-congruency on the current trial , location-congruency on the current trial , direction-congruency on the preceding trial , and location-congruency on the preceding trial  were conducted on the mean RTs and error proportions.F = 5.8; p < 0.04; MSE = 310.1, and when the current trial was associated with an incongruent as compared to a congruent stimulus location (388 vs. 362 ms), F = 39.1; p < 0.01; MSE = 1034.2. Within-dimension Gratton effects reliably occurred for both distractor dimensions. The direction-based congruency effect was reduced from 17 to -6 ms after a direction-incongruent trial as compared to a direction-congruent trial, F = 7.9; p < 0.02; MSE = 858.7, and the location-based congruency effect was reduced from 57 to −4 ms after a location-incongruent trial as compared to a location-congruent trial, F = 125.6; p < 0.01; MSE = 443.5. Regarding between-dimension sequential modulations, the direction-based congruency effect was reduced after a location-incongruent trials as compared to a location-congruent trials (0 vs. 11 ms), F = 6.9; p < 0.02; MSE = 257.9. Likewise, the location-based congruency effect was reduced after direction-incongruent as compared to after direction-congruent trials (24 vs. 29 ms), F = 4.7; p < 0.05; MSE = 87.2.Responding took longer when the current trial was associated with an incongruent as compared to a congruent arrow direction (378 vs. 372 ms), F = 3.2; p = 0.10; MSE = 0.00116. Errors were more frequent, however, when the current trial was associated with an incongruent as compared to a congruent stimulus location (5.6 vs. 3.4%), F = 6.9; p < 0.03; MSE = 0.00434. In addition, an incongruent arrow direction on the preceding trial reduced errors from 4.9 to 4.1%, F = 5.2; p < 0.04; MSE = 0.00061. Again, within-dimension Gratton effects occurred for both dimensions: The direction-based congruency effect was reduced from 1.9 to −0.3% after a direction-incongruent trial compared to a direction-congruent trial, F = 8.4; p < 0.02; MSE = 0.00098, and the location-based congruency effect was reduced from 7.4 to -3.0% after a location-incongruent trial compared to a location-congruent trial, F = 25.4; p < 0.01; MSE = 0.00630. By contrast, no between-dimension sequential modulation occurred, both Fs < 1.In the error analysis, the main effect of direction-congruency failed to reach significance , N − 1) → direction-congruency (N)Location-congruency , location-congruency in trial N − 1 , and abstract correspondence . Figure F = 4.9; p < 0.05; MSE = 207.4, and F = 5.7; p < 0.04; MSE = 139.6, respectively. As predicted by the semantic generalization hypothesis, this was modulated by the abstract correspondence, F = 10.3; p < 0.01; MSE = 31.1. Whereas on abstract match trials, the direction-based congruency effect amounted to 14 ms after location-congruent trials and −3 ms after location-incongruent trials, abstract mismatch trials were associated with 8 and 4 ms of direction-based interference after location-congruent and location-incongruent trials, respectively. The error data yielded no significant effects.N − 1) → Location-congruency (N)Direction-congruency , direction-congruency in trial N − 1 , and abstract correspondence . Figure F = 35.8; p < 0.01; MSE = 564.0, and, F = 4.9; p < 0.05; MSE = 29.6, respectively. In line with the semantic generalization hypothesis, reduction of the location-based congruency effect after direction-incongruent trials occurred selectively on abstract match trials, F = 6.2; p < 0.03; MSE = 131.4. Specifically, on abstract match trials, the location-based congruency effect amounted to 35 ms after direction-congruent trials and 20 ms after direction-incongruent trials, whereas on abstract mismatch trials the location-based congruency effect was 21 ms after direction-congruent trials and 28 ms after direction-incongruent trials. The error data yielded only a significant main effect of location-congruency, F = 7.4; p < 0.02; MSE = 0.00221.Experiment 1 replicated previously found sequential modulations of location-based interference and of direction-based interference. Although these within-dimension Gratton effects are in line with the conflict adaptation hypothesis, they can also be accounted for in terms of distractor-target or distractor-response feature integration because of the complete confound of the sequences of congruency levels and the sequences of specific distractor and target/response conjunctions. In addition, replicating the finding of Kunde and Wühr , both diAlthough we intermixed blocks of trials in which participants responded to the direction of the stimulus arrow, direction-based interference  was overall small–considerably smaller than location-based interference and smaller than in the study of Kunde and Wühr . Given tIn Experiment 2 participants responded to a triangle that pointed either to the left or to the right by pressing the spatially corresponding key. Directional distractor information was presented in the form of a pair of different triangles, in which the target stimulus was embedded, forming a vertical target-flanker configuration. Like in Experiment 1, the stimuli could be presented in a left-sided or in a right-sided screen location. Experiment 2 thus combined location-based interference with interference evoked by spatially adjacent flanker stimuli. Noteworthily, previous studies that combined location-based interference and flanker interference, however in the context of non-spatial tasks, failed to obtain between-dimension Gratton effects  to the left or to the right of the screen center, and extending 5.5 cm vertically and 1.7 cm horizontally. The central (target) triangle was presented on the horizontal midline. Triangles had a side length of 1.8 cm and could point to the left or to the right. The same response keys were used as in Experiment 1.The procedure of Experiment 2 was the same as the procedure of Experiment 1 with the following exceptions. First, participants responded to the pointing direction of the central triangle by pressing the spatially corresponding response key. Second, on each trial, the pointing direction (left vs. right) of the central triangle as well as the pointing direction of the flanker triangles (left vs. right) were chosen randomly with the only constraint that the two distractor triangles pointed in the same direction. Figure The same analyses and exclusion criteria were applied as in Experiment 1. RT outlier exclusion eliminated 0.04% of the data.F = 296.7; p < 0.01; MSE = 996.4. In contrast, there was no overall location-based congruency effect, F < 1. Direction- and location-based interference interacted however, yielding a location-based congruency effect of 11 ms on direction-congruent trials and a slightly reversed location-based congruency effect of −5 ms on direction-incongruent trials, F = 19.4; p < 0.01; MSE = 199.5. Within-dimension Gratton effects occurred for both dimensions. The direction-based congruency effect was reduced from 82 to 59 ms after a direction-incongruent compared to after a direction-congruent trial, F = 32.9; p < 0.01; MSE = 252.6, and the location-based congruency effect was reversed from 37 ms after location-congruent to −31 ms after location-incongruent trials, F = 98.0; p < 0.01; MSE = 715. The latter effect was further modulated by an interaction with direction-congruency of the preceding trial, F = 6.6; p < 0.03; MSE = 212.4, indicating that the sequential modulation was somewhat larger when the preceding trial involved incongruent flankers. The direction-based congruency effect was also reduced after location-incongruent trials compared to after location-congruent trials (65 vs. 76 ms), F = 16.0; p < 0.01; MSE = 113.4. This between-dimension Gratton effect was further modulated by an interaction with location-congruency of the current trial, F = 39.2; p < 0.01; MSE = 148.1, indicating that the effect was confined to location-congruent trials. The location-based congruency effect was not affected by the direction-congruency level of the preceding trial , F = 1.4; p = 0.25; MSE = 65.6.Responses were slower when the current trial was associated with incongruent as compared to congruent flankers (486 vs. 415 ms), F = 28.5; p < 0.01; MSE = 0.00163, and with incongruent than with congruent stimulus locations (3.5 vs. 1.9%), F = 12.9; p < 0.01; MSE = 0.00116, and less frequent after a trial with incongruent than congruent flankers (2.4 vs. 3.0%), F = 5.5; p < 0.04; MSE = 0.00042, as well as after a trial with an incongruent than with a congruent stimulus location (2.0 vs. 3.5%), F = 14.9; p < 0.01; MSE = 0.00087. Direction- and location-based interference interacted overadditively, F = 5.3; p < 0.04; MSE = 0.00068. Both the direction-based and the location-based congruency effect were reduced after a location-incongruent compared to a location-congruent predecessor trial, 1.9 vs. 3.6%, F = 6.2; p < 0.03; MSE = 0.00075, and −1.4 vs. 4.5%, F = 30.6; p < 0.01; MSE = 0.00172, respectively. A three-way interaction involving location-congruency of the current and of the preceding trial and direction-congruency of the current trial, F = 13.3; p < 0.01; MSE = 0.00062, indicated that the location-based Gratton effect was more pronounced on trials with incongruent than with congruent flankers. Neither direction- nor location-based interference was affected by the direction-congruency level of the preceding trial, both Fs < 1.In the error analysis, all main effects were significant, indicating that errors were more frequent with incongruent than with congruent flankers (4.1 vs. 1.3%), N − 1) → direction-congruency (N)Location-congruency , location-congruency in trial N − 1 , and abstract correspondence . Figure F = 294.2; p < 0.01; MSE = 504.2, and F = 14.7; p < 0.01; MSE = 85.6, respectively. Abstract mismatch trials were associated with an increase in overall RTs of 8 ms, F = 22.1; p < 0.01; MSE = 95.5, and with an increase in direction-based interference of 8 ms, F = 5.4; p < 0.04; MSE = 108.5. There was no sign of a three-way interaction involving direction-congruency of the current trial, location-congruency of the preceding trial, and abstract correspondence, F = 1.2; p = 0.30; MSE = 32.1. The error analysis replicated the main effects of direction-congruency and preceding location-congruency as well as the interaction between these two factors from the overall analysis, F = 30.0; p < 0.01; MSE = 0.00077, F = 13.4; p < 0.01; MSE = 0.00045, and F = 7.2; p < 0.02; MSE = 0.00033. Mirroring the RT results, the reduction of the direction-based congruency effect after location-incongruent trials was not modulated by abstract correspondence, F < 1.N − 1) → Location-congruency (N)Direction-congruency , direction-congruency in trial N − 1 , and abstract correspondence . Figure F = 11.9 p < 0.01; MSE = 141.2, indicating that abstract mismatch trials were associated with longer RTs than abstract match trials (453 vs. 446 ms). In addition, incongruent flankers in the preceding trial slowed responding on abstract match trials by 12 ms and speeded up responding on abstract mismatch trials by 10 ms, F = 31.7; p < 0.01; MSE = 110.0. As in the overall analysis, the location-based congruency effect was not significantly affected by the direction-congruency level of the preceding trial, F = 3.1; p = 0.10; MSE = 44.4. However, there was a significant three-way interaction, F = 14.7; p < 0.01; MSE = 83.7. As can be seen in Figure F = 13.6; p < 0.02; MSE = 0.00060. Also, the main effect of direction-congruency on the preceding trial approached significance, F = 3.8; p = 0.07; MSE = 0.00024. Contrary to the RT analysis, incongruent flankers in the preceding trial decreased the error rate on abstract match trials by 1.7% and increased the error rate on abstract mismatch trials by 0.6%, F = 15.8; p < 0.01; MSE = 0.00025. Although there was no overall reduction of location-based interference after direction-incongruent trials, F < 1, a significant three-way interaction indicated that the location-based congruency effect was larger after direction-congruent than direction-incongruent trials on abstract match trials, whereas it was larger after direction-incongruent than after direction-congruent trials on abstract mismatch trials, F = 8.2; p < 0.02; MSE = 0.00033.Barring minor procedural differences Experiment 2 differed from Experiment 1 in that the target stimulus features were perceptually similar to the direction distractor features. As expected, the direction-based congruency effect was considerably larger under these conditions than in Experiment 1. In contrast to this enhancement of distractor interference, the main effect of location-based interference failed to reach significance in RTs.Despite the absence of a significant overall effect of location-based interference, however, a clear-cut pattern of sequential modulations of congruency effects occurred. Again, we found within-dimension Gratton effects for both distractor dimensions, which cannot unequivocally be attributed to conflict adaptation because of the confound with distractor-target or distractor-response sequences. Replicating the findings of Experiment 1, a between-dimension Gratton effect was found for direction-based interference, and—deviating from the results of Experiment 1—displayed comparable strength for abstract match and mismatch trials. As such, these findings seem to provide novel evidence for the conflict adaptation account. However, the modulations of location-based interference pose a problem for this interpretation. Specifically, although there was no overall modulation of the location-based congruency effect by the direction-based congruency level of the preceding trial, this absence resulted from a between-dimension Gratton effect on abstract match trials and a reversed between-dimension Gratton effect  on abstract mismatch trials. Viewed from the conflict adaptation perspective, the latter effect would suggest that location-congruent rather than location-incongruent trials increased attentional focusing. There seems to be no straightforward reason for such an assumption.By contrast, the semantic generalization hypothesis offers a plausible explanation for the co-occurrence of a between-dimension Gratton effect for one of the distractor dimensions and a reversed between-dimension Gratton effect for the other distractor dimension, on abstract mismatch trials. As noted in the Introduction, on abstract mismatch trials conjoined repetitions/alternations regarding the response and the location-to-direction sequence are associated with partial repetitions regarding the response and the direction-to-location sequence and vice versa. We argued that, in the case that both abstract feature sequences yield equally strong effects, this arrangement should result in a null effect on abstract mismatch trials, as found in Experiment 1. On the other hand, there is no a priori reason to assume that the activation of a stored location code by perceiving a corresponding arrow direction is about as strong as the activation of a stored direction code by perceiving a stimulus in a corresponding location. Therefore, it is also conceivable that one of the assumed mechanisms, activation of a location code by a perceived direction or activation of a direction code by a perceived location, is substantially stronger than the other one, depending on yet unidentified stimulus and task characteristics.Consider the case that the activation of a location code elicited by perceiving a corresponding arrow direction is stronger than the activation of a direction code elicited by perceiving a corresponding stimulus location. For abstract match trials, in which effects of the location-direction sequence and the direction-location sequence work in the same direction, this would lead to qualitatively the same expectation as an equal strength assumption. The prediction would be different, however, for abstract mismatch trials. Performance on these trials should be more affected by the location-direction sequence than by the direction-location-sequence. Looking at the abstract mismatch trials in Table Regarding location-based interference after direction-congruent and direction-incongruent trials, however, the assumption that direction-location sequences are less influential than location-direction sequences would lead to the opposite prediction for abstract mismatch trials, that is increased location-based interference after direction-incongruency, as compared to after direction-congruency. This is because on such trials between-dimension congruency level repetitions are associated with partial repetitions regarding the abstract location-direction feature and the response, whereas between-dimension congruency level alternations are associated with partial repetitions regarding the abstract location-direction feature and the response. , reduced direction-based interference after location-incongruent trials and increased location-based interference after direction-incongruent trials (as found in Experiment 2), or reduced location-based interference after direction-incongruent trials and increased direction-based interference after location-incongruent trials, depending on the relative strengths of “location by direction” and “direction by location” activation. The results of Experiment 2 are consistent with the semantic generalization hypothesis if it is assumed that activation of a location code by perceiving a corresponding arrow outweighs activation of a direction code by perceiving a corresponding stimulus location under the conditions set up in the experiment.The present article deals with trial-to-trial modulations of interference evoked by processing a distractor stimulus feature, focusing on reductions of interference evoked by one distractor dimension after conflict evoked by a perceptually different distractor dimension. The presence or absence of such between-dimension Gratton effects bears theoretical importance for two reasons. First, the specific pattern of distractor dimensions for which between-dimension Gratton effects are found may provide insights regarding the specific processes of conflict detection and adaptation. Second, findings of between-dimension Gratton effects with an experimental set-up like the one realized in the current study might provide valuable evidence for the notion of attentional conflict adaptation as such. This is because so far within-dimension Gratton effects could not be replicated in conditions of rigorous control of feature sequence and distractor-target/response contingencies unless the task used was divided into a pair of non-overlapping two-choice tasks  accords with the assumption that semantic overlap between distractor dimensions plays a facilitative role in generating such effects. It is conceivable that semantic overlap of distractor dimensions promotes representing both dimensions in a linked structure, thereby possibly enhancing the likelihood of generalized conflict adaptation. Although we cannot dismiss this possibility, an alternative explanation seems better suited to account for the overall pattern of results we obtained. Specifically, we hypothesized that overlap between abstract stimulus features may result in activation of a stimulus feature code in episodic memory by perception of a semantically related feature of the other dimension. With this assumption it is possible, in principle, to account for between-dimension Gratton effects in terms of processing disadvantages on between-dimension congruency level alternations due to partial repetitions regarding the responses and abstract stimulus features.Consistent with this hypothesis, the between-dimension Gratton effects in Experiment 1 were confined to abstract match trials, whereas there was no trial-to-trial modulation of the congruency effect on abstract mismatch trials, a result difficult to explain by the assumption of (dimension-unspecific) conflict adaptation. In Experiment 2, the task required responding to the direction of a target stimulus. Although this manipulation did not alter the sequences of concrete and abstract distractor and response features, it added perceptual overlap of the target stimulus dimension with the direction distractor dimension . This arrangement resulted not only in an overall larger congruency effect evoked by the direction distractor but also yielded a more complicated pattern of between-dimension Gratton effects. Precisely, whereas a between-dimension Gratton effect, unaffected by the abstract feature sequence, occurred for the direction dimension, the location dimension was associated with a between-dimension Gratton effect for abstract match trials but a reversed Gratton effect for abstract mismatch trials. Viewed from a conflict adaptation perspective, this pattern of results would suggest that attentional focusing was stronger after (direction-) congruent than after (direction-) incongruent trials on abstract mismatch trials. There seems to be no straightforward reason for this assumption. By contrast, the semantic generalization hypothesis offers a plausible explanation for this pattern of findings.Specifically, considering the possibility of different strengths of abstract feature sequence effects on location-direction and direction-location transitions, three different patterns of a three-way interaction of congruency of the current trial, congruency regarding the other distractor dimension on the preceding trial, and the abstract feature sequence (match vs. mismatch) are conceivable. The results obtained in Experiments 1 and 2 constitute one of them each. Given the absence of an independent measure of relative strength of abstract feature sequence effects, this interpretation can only be applied with caution. On the other hand, because all of the three possible result patterns involve a modulation of the sequential congruency effect by the sequence of abstract stimulus and response features, they seem difficult to account for in terms of conflict adaptation. Further research regarding semantic generalization of feature integration effects could be undertaken by means of manipulating semantic overlap between prime and probe stimuli in Hommel's feature integration paradigm (e.g., Hommel, As noted in the Introduction, recent studies have provided strong evidence for conflict adaptation, uncontaminated by feature repetitions and biased distractor-target/response contingencies. Investigating sequential congruency effects evoked by different perceptual dimensions complement this approach and seem particularly useful to examine the question of specificity vs. generality of conflict adaptation. Providing initial evidence for semantic generalization effects of stimulus and response feature integration, however, our current study demonstrates a limitation in attributing between-dimension Gratton effects to generalized conflict adaptation if the two distractor dimensions overlap semantically. Such inference seems justified only if between-dimension Gratton effects are consistently found not only for abstract match trials but also for abstract mismatch trials, for both distractor dimensions that are combined in the experiment.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In order to improve the homogeneity and dispersity of the sericite in methylphenyl-silicone resin, the agglomerate state of the sericites was controlled effectively. The dispersive model of the sericite in methylphenyl-silicone resin was designed also. First, the modified sericite was prepared using hexadecyl trimethyl ammonium bromide as the intercalating agent. Then, functional sericite was incorporated into methylphenyl-silicone by terminal hydroxyl. The structure and dispersive performance of the hybrid polymers was charactered by analytical instruments. Scanning electron microscopy and Transmission electron microscope, Laser scanning confocal microscope and X-ray diffraction analysis showed that functional sericite was dispersed homogeneously in methylphenyl-silicone resin matrix. X-ray photoelectron spectroscopy analysis showed that the absorption peaks of the Si-OH band of methylphenyl-silicone resin were decreased and the Si-O-Si band was increased. This change evidently showed a significant role to enhance the reaction degree of the functional sericite in methylphenyl-silicone resin. In the current study, nanometre-scale, self-assembly ,2 and suSilicone resin matrix composites, as a kind of heat-resistant material, have many excellent dielectric properties, low heat expansion coefficient and light weight, so that silicone resin is a promising coating material, it has widely applied on electrical equipment, planes and vehicle so on . The syn2[AlSi3O10](OH)2, Si-O tetrahedron structure of the sericite and Si-O backbone structure of the molecular of the silicone resin is similar. If the dispersion of sericite in the silicone resin can improve, the heat stability of the silicone resin can enhance. The aim of this study is develop a dispersive strategy of sericite in methylphenyl-silicone resin and designed the dispersive model.The chemical formula of the sericite is KAl2SO4 (98%), 0.5 g NaNO3 and 3 g of KMnO4) at ice-bath, the temperature of the mixture was maintained below 20°C. The mixture was then stirred at 35°C for 120 min, 50 mL distilled water was slowly added, the temperature of the reaction solution was rises to 98°C for 30 min. The reaction was terminated by 100 mL distilled water followed by 1 mL H2O2 solution. The sericites were separated by centrifugation and washed by 10 ml 10% HCl solution, and then the sericites were collected using freezing dry for 24 h.The sericites and methylphenyl-silicone resin were obtained from Chuzhou and Shanghai Hersbit Chemical Co., Ltd, China, respectively. The crude sericites were exfoliated using chemical method. 1 g sericites were added gradually to mixed solution  were loaded into a glass container. The mixture was sonicated for 6 h at room temperature. The solution was separated in a centrifuge giving rise to functional sericites sheets. Then, the sericites sheets were added the saturated NaCI solution (concentration 10%) by ion exchange. It was separated by centrifugation, washed repeatedly by deionized water until chlorides could not be detected. Finally, the product was obtained by freezing dry for 24 h.Active surface reagent- hexadecyl trimethyl ammonium bromide  was added to 100 mL butyl alcohol, 1 g sericites were added gradually. The mixture was then stirred at 90°C for 8 h. The modified sericites were separated by centrifugation, and then above product was dried in a vacuum oven at 60°C.The modified sericite and methylphenyl-silicone resin was mixed by the ultrasonic at -10°C-5°C ranges 2 h. The doping content of the modified sericite was 2% weight percent in methylphenyl-silicone matrix. Above mixture was poured into teflon molds, gradually heated at 80°C, 100°C, 120°C, 150°C, 180°C, 200°C and 250°C for the duration of 1 h. The modified sericite / methylphenyl-silicone resin could be obtained after the completion of the curing processes.-1 using KBr disks. X-ray diffraction (XRD) data were collected with a Shimazdu X-ray diffractometer equipped. X-ray photoelectron spectroscopy (XPS) analysis of the samples was carried out using a VG electron spectrometer . Thermogravimetric analysis (TG) was performed on a NETZSCH thermoanalyzer .Morphological features of the modified sericite / methylphenyl-silicone resin were observed by a high-resolution scanning electron microscopy (SEM) . Fourier-transformed infrared (FT-IR) spectrometer  was operated in IR region from 400 to 4000 cmd-values are increased 0.14, 0.06 and 0.04, respectively. Furthermore, the size distribution of the sericite is shown in The provided SEM images of the sericites shown in -1 and 1160–1000 cm-1, respectively. The absorption peaks of the Si-OH groups at 3620 cm-1 are decreased, observably. The observed phenomenon suggests that the condensation reaction of the Si-OH group had taken place between the modified sericites and the methylphenyl-silicone. The other change can be observed for the peak at 1160–1000 cm-1. The intensity of the Si-O-Si absorption band is increased with the incorporation of modified sericites. These results further confirm that the modified sericites is indeed incorporated into the methylphenyl-silicone resin rather than as a mixture. When curing temperature increased , the intensity of the Si-O-Si absorption peak increased and the Si-OH absorption peak decreased for the modified sericite / methylphenyl-silicone resin from The reaction process of the modified sericite / methylphenyl-silicone resin is given in 2p and O1s peaks. 2p and O1s peaks for the methylphenyl-silicone and modified methylphenyl-silicone at 250°C. These peaks are centered at 103 eV which corresponds to C-Si-O1.5, at 102 eV which corresponds to Si-OH, in the methylphenyl-silicone chain and modified sericite. In 1.5 is increased while that of Si-OH is decreased. Similarly, The high resolution XPS survey spectra showed distinct carbon, silicon and oxygen peaks, representing the major constituents of the methylphenyl-silicone resin are investigated . Table 1The collected data by XRD clearly shows that the modified sericite was exfoliated at a curing temperature of 250°C . After tThe white areas in the SEM images represent the sericite and the black areas are the methylphenyl-silicone resin . The imaIf the interference and entanglements has intermolecular or intramolecular, polymer molecular chain did not free stretching, this will affect flexibility of molecular chain. Sketch is shown in Preparation of the functional sericite /methylphenyl-silicone resin composites was investigated. The modified sericite was dispersed homogeneously in methylphenyl-silicone resin matrix. Morphological features and molecular structure of product is characterizated by SEM, TEM, LSCM, XPS, XRD. From above analysis, the dispersity models of functional sericite / methylphenyl-silicone resin were designed. The study achievements have great significance in development of silicone resin composites.S1 MaterialsFigure A in S1 Materials), CH3 and CH2 absorption bands is shown as 2920 cm-1and 2850 cm-1. This result shows that the hexadecyl trimethyl ammonium bromide was on the surface of the sericite or went into the interbedded structure of the sericite. After the modification, the sericite was changed from the hydrophile to the lipophilic compound. From FT-IR analytical result, it can be also deduced; crude sericite has been activated (see S1 Materails).The modified process of the sericite and modified sericite is shown in the Supporting Information. FT-IR spectra are presented ((DOC)Click here for additional data file.


Nanoparticles have attracted increasing attention for local drug delivery to the inner ear recently. Bovine serum albumin (BSA) nanoparticles were prepared by desolvation method followed by glutaraldehyde fixation or heat denaturation. The nanoparticles were spherical in shape with an average diameter of 492 nm. The heat-denatured nanoparticles had good cytocompatibility. The nanoparticles could adhere on and penetrate through the round window membrane of guinea pigs. The nanoparticles were analyzed as drug carriers to investigate the loading capacity and release behaviors. Rhodamine B was used as a model drug in this paper. Rhodamine B-loaded nanoparticles showed a controlled release profile and could be deposited on the osseous spiral lamina. We considered that the bovine serum albumin nanoparticles may have potential applications in the field of local drug delivery in the treatment of inner ear disorders. Inner ear disorders, including sensorineural hearing loss (SSHL), commonly occur in clinics. The traditional systemic therapies are almost ineffective due to the blood-labyrinth barrier, which prevents the transport of drugs from the serum. Local drug delivery, especially intratympanic injection, has become popular for two decades because of its efficiency and safety. The round window membrane (RWM) is a semipermeable membrane between the middle and the inner ear, through which particles less than 3 μm in diameter could penetrate.Local drug delivery to the inner ear by intratympanic injection was first described by Schuknecht in 1956 in the treatment of Ménière's disease.l-glutamic acid) particles could be released in a sustained manner with maintained biological activity and efficiently rescue primary auditory neurons in the cochlea of guinea pigs with sensorineural hearing loss[Although intratympanic injection is easy to perform in the clinic, the loss of drug through the Eustachian tube becomes the obstacle to treat inner ear disorders efficiently. Thus, hydrogel- and particle-based vehicles (or carriers) have been investigated recently for sustained and prolonged drug supply. In 1998, Balough et al. described that the local injection of a fibrin-based sustained release vehicle impregnated with gentamicin allowed for a prolonged effect without absorption in the untreated ear or blood. Horie eing loss. NowadayVarious methods such as desolvation, emulsion, template, microfluidic, mechanical stretching, and self-assembly have been reported to prepare protein-based particles or capsules-11. In tin vivo studies. In vivo biodistribution suggested that the RhB released as well as the RhB-loaded BSA-NPs (RhB-BSA-NPs) tended to accumulate and penetrate through the RWM of guinea pigs. Therefore, the BSA-NPs would be prospectively considered as controlled release carriers for local drug delivery in the treatment of inner ear disorders.BSA is a natural protein able to form complexes in various shapes. This protein is biocompatible, biodegradable, nontoxic, and nonimmunogenic. Due to these features, albumin particles are a good system for drug and antigen delivery-14. To tBSA and RhB were purchased from Sigma-Aldrich . Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technology Inc. . Ultrapure water used in all experiments was produced by Milli-Q synthesis system . L929 mouse fibroblast cells  were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  containing 10% fetal bovine serum (FBS) at 37°C with 5% CO2. Guinea pigs weighing 250 ~ 300 g were purchased from the Tianjin Experimental Animal Center, People's Republic of China, and had free access to food and water. Animal study protocols were approved and performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals.BSA-NPs were prepared by the desolvation method. Briefly described, 100 mg of BSA was dissolved in 1 ml of sodium chloride solution (10 mM). Then, 8.0 ml of ethanol was added dropwise into the BSA solution under magnetic stirring (400 rpm) at room temperature. Subsequently, the as-prepared BSA-NPs were cross-linked with 0.2% glutaraldehyde (GA) for 24 h or denatured at 70°C for 30 min. BSA-NPs (50 mg) were incubated with certain amounts  of RhB for 2 h in the preparation of RhB-BSA-NPs. The particles were centrifuged and washed with ultrapure water.The morphological characteristics were determined by transmission electron microscopy , scanning electron microscopy , and confocal laser scanning microscopy . For TEM, a drop of diluted suspension of BSA-NPs was placed on the copper grid and the air-dried specimen was observed. For SEM, a drop of diluted suspension was deposited on a silicon wafer. The air-dried sample was coated with gold and observed. RhB-BSA-NPs were observed by CLSM at an excitation wavelength of 555 nm and an emission wavelength of 580 nm.The BSA-NPs were dispersed in ultrapure water at a concentration of 0.1 mg/ml. The particle size and zeta potential determinations were performed by using a Malvern particle size analyzer .BSA-NPs (50 mg) were incubated with RhB (5 ~ 20 mg) for 2 h. After washing with ultrapure water, the supernatants were collected and analyzed for residual drug concentration by UV-vis analysis.The drug loading capacity and encapsulation efficiency were calculated as follows:w / w%) = amount of RhB in BSA-NPs/RhB initially added × 100Encapsulation efficiency . The amount of RhB released was evaluated at a series of time points, and the release curve was made accordingly.Cells were seeded in 96-well plates at a density of 1,000 cells/well. BSA-NPs with GA fixation (NP-GA) or heat denaturation (NP-H) were added to each well for a 24-h incubation. Cell viability was determined by CCK-8 assay. Untreated cells served as the control. The morphology of L929 cells in each group was also observed by using a phase contrast microscope.Guinea pigs were killed to sample the acoustic bullae (including the RWM). The acoustic bullae were placed in the solution of BSA-NPs and shaking for 30 min at 37°C. The air-dried specimens were observed by SEM.In vivo imaging system  was used to trace the particles at time points of 0 and 72 h. The RWM was then imaged by fluorescence microscopy and SEM to observe the distribution of RhB and BSA-NPs.The penetration of RhB released from the RhB-BSA-NPs was evaluated by live images and microscopes. Guinea pigs were anaesthetized and the RWMs were exposed. The heat-denatured RhB-BSA-NPs and RhB dispersed in PBS were injected slowly into the bullae of the right and left ear, respectively. The left ear injected with RhB solution was the control. t test was used in SPSS 12.0 to determine significant differences between groups, and P values less than 0.05 were considered statistically significant.The statistical data was presented as the mean value and standard deviation. The analysis of BSA-NPs were prepared by the desolvation method in high yield (about 95%). Figure Rhodamine B was used as a model drug for observation and evaluation of drug loading capacity. The morphology and structure of RhB-loaded NP-H Figure a did notThe BSA-NPs and RhB-BSA-NPs had zeta potential values of -15.4 and +4.98 mV, respectively. The potential difference demonstrated that the positively charged RhB had an interaction with the negatively charged BSA-19.The drug loading capacity and encapsulation efficiency of BSA-NPs were also evaluated. The drug loading capacity of BSA was 15.4% for RhB Figure c. The main vitro drug release profile of RhB from BSA-NPs is shown in Figure in vitro.The In vitro experiment of BSA-NPs cross-linked with GA or denatured by heat against L929 cell lines were performed by CCK-8 to evaluate the cytocompatibility. As shown in Figure P = 0.001) than that of the control possibly because the water wash in this study was only once. These results indicated that the NP-H had a better cytocompatibility than the NP-GA. The slight cytotoxicity of NP-GA was in agreement with that reported by Speer[P = 0.114) and the control.by Speer. There wThe shape of L929 cells incubated with NP-H maintained high viability after the assay Figure d while rAs for the good cytocompatibility, BSA-NPs with heat denaturation were loaded with RhB and used to evaluate the local drug delivery. Acoustic bullae of guinea pigs with entire RWM were isolated and injected with RhB-BSA-NP (right ear) and RhB solution (left ear). The live images were taken immediately Figure a. Three The guinea pigs were then killed and the temporal bones and RWMs were separated. The nanoparticles still attached on the RWM Figure a. The SEIn summary, BSA-NPs were fabricated via a desolvation method. The heat-denatured BSA-NPs had a great potential application for local drug delivery into the cochlea to treat inner ear diseases due to the tiny size, good biocompatibility, drug loading capacity, and controlled release profile. Further studies will focus on the evaluation of drug-loaded BSA-NPs, including prednisolone. We will evaluate their pharmacokinetics, pharmacodynamics, and delivery mechanism in animal model. The BSA-NPs also shed light in the treatment of human inner ear diseases.BSA: bovine serum albumin; BSA-NP: bovine serum albumin nanoparticles; RWM: round window membrane; PLGA: polylactic/glycolic acid; RhB: rhodamine B; RhB-BSA-NP: RhB-loaded BSA-NP; GA: glutaraldehyde; CCK-8: cell counting kit-8; NP-GA: BSA-NPs with GA fixation; NP-H: BSA-NPs with heat denaturation; DLS: dynamic light scattering; TEM: transmission electron microscope; SEM: scanning electron microscopy.The authors declare that they have no competing interests.ZY, ZZ, GH, QX, and MY performed the experiments and analyzed the results. ZY and MY conceived and designed the experiments, analyzed the results, and participated in writing the manuscript. All authors read and approved the final manuscript.ZY is a professor from the Department of Otorhinolaryngology, The Second Artillery General Hospital of Chinese People's Liberation Army, Beijing, 100088, People's Republic of China, and Center of Otorhinolaryngology, Naval General Hospital of Chinese People's Liberation Army, Beijing, 100037, People's Republic of China. MY is a Ph.D. from the Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, College of Basic Medicine, China Medical University, Shenyang 110001, People's Republic of China. ZZ, GH, and QX are Ph.D. from the Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, The Key Laboratory of Biomedical Material of Tianjin, Tianjin, 300192, People's Republic of China.


Cell proliferation was observed to be inhibited subsequent to transfection with miR-206. It was suggested that miR-206 induced cell cycle G1/S phase arrest by suppressing the expression of cyclinD2. The results of the present study concluded that miR-206 inhibits glioma progression via the regulation of cyclinD2 and that miR-206 may be a novel biomarker with potential for use as a therapeutic target in gliomas.MicroRNAs are short single-stranded non-coding RNA molecules that function as regulators of tumor progression, including regulation of glioblastoma multiforme, which is a World Health Organization grade IV glioma. Based on the results of a microRNA microarray, which included 198 patients with glioma from the Chinese Glioma Genome Atlas data set, it was observed that microRNA-206 (miR-206) was downregulated in high-grade (grades III and IV) gliomas compared with grade II gliomas. In addition, high expression of miR-206 was associated with longer overall survival time in glioma patients. The present study aimed to investigate the biological functions of miR-206 in glioma progression  Glioblastoma multiforme (GBM) is the most prevalent lethal intracranial tumor in adults . It is cMicroRNAs are small non-coding RNA molecules that are comprised of 16~22 nucleotides and downregulate translation by targeting mRNAs . These mPrevious studies reported that microRNA-206 (miR-206) expression was markedly reduced in osteosarcoma and lung cancer, and that it was necessary for cell growth, migration, apoptosis and invasion ,9. In huHowever, the function of miR-206 in gliomas remains to be fully elucidated. In the present study, the aim was to investigate the functional role of miR-206 in gliomas and to further elucidate the mechanism of miR-206 in tumorigenesis and progression. The present study hypothesized that miR-206 acts as a tumor suppressor and suppresses glioma cell proliferation via cyclinD2.www.cgga.org.cn/). The microRNA microarray analysis was conducted on 198 patients with glioma and based on the gene expression microarray and cyclinD2 expression was analyzed in 225 patients with grade II glioma or high-grade gliomas (HGGs), of which 158 patients also underwent microRNA microarray analysis. In the present study, the patients underwent a resection operation between January 2006 and December 2010 and subsequently received adjuvant treatment of temozolomide combined with radiotherapy. The present study was approved by the Beijing Tiantan Hospital  institutional review board and written informed consent was obtained from all patients.All patient information was obtained from the Chinese Glioma Genome Atlas -miR-206 mimics were synthesized by Guangzhou RiboBio Co., Ltd. . The following primer sequence was used in the present study: UGGAAUGUAAGGAAGUGUGUGG. Cells were cultured at a density of 8×104 cells/well in six-well plates. miR-NC and hsa-miR-206 oligosaccharides were transfected into LN229 cells at a final concentration of 50 nmol/l using riboFECTTM CP reagent  according to the manufacturer’s instructions. The miR-NC contained a scrambled sequence for the control.The human LN229 GBM cell line was purchased from the Chinese Academy of Sciences Cell Bank . LN229 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% glutamine . All cells were incubated at 37°C in an atmosphere supplemented with 5% CORenilla luciferase activity using the Dual Luciferase Reporter Assay System  in accordance with the manufacturer’s instructions. Renilla luciferase was used for normalization.For the luciferase assays, the luciferase reporter vector cyclinD2-3′UTR and the negative control were specifically synthesized by Shanghai GenePharma Co., Ltd. . The cells were cultured in 12-well plates and transfected with hsa-miR-206 and co-transfected with the luciferase reporter vector. Following incubation for 48 h, cells were harvested and analyzed for ®-Multi Detection system; Promega Corp.), which were proportional to the number of live cells.Cell proliferation was assessed using MTT  and colony formation assays. Subsequent to transfection with miR-NC and hsa-miR-206 oligosaccharides for 24 h, LN229 cells were transplanted at a density of 2,000 cells/well with five replicated wells for each group in the 96-well plates. In order to determine relative cell growth, MTT assays were conducted for five consecutive days by adding 20 μl MTT solution (5 mg/ml) to each well and incubating at 37°C for 4 h. To solubilize formazan crystals, 150 μl DMSO (Sigma-Aldrich) was added and the absorbance values were detected at 490 nm using a microplate reader  and stained with crystal violet (Sigma-Aldrich). All colonies were manually counted by using a microscope .Western blot analysis was conducted to assess cyclinD2 expression in transfected cells. The total proteins were isolated from the LN229 cell lines transfected with negative control plasmids and miR-206 mimics for 48 h. These cells were isolated with trypsin-EDTA , and lysed in lysis buffer  for a minimum of two hours on ice. Subsequent to quantification of protein, an equal amount of protein (10 μg) was added into the sample wells, separated using 10% SDS-PAGE (Sigma-Aldrich) and transferred to polyvinylidene difluoride membranes . Western blotting was conducted with monoclonal rabbit anti-cylinD2 immunoglobulin G (IgG) as the primary antibody  incubated at 4°C overnight. Monoclonal rabbit anti-GAPDH IgG antibody, incubated at a dilution of 1:1,000 at 4°C overnight  was used to ensure equal protein loading. The secondary antibodies were horseradish peroxidase-conjugated anti-rabbit IgG, diluted at 1:5,000 and purchased from Origene 4) treated with negative control and miR-206 mimics were plated in six-well plates. Subsequent to incubation for 48 h, the cells were collected by trypsinization, fixed for 24 h in 70% ethanol  and stained with propidium iodide Beyotime Institute of Biotechnology, Haimen, China) for 30 min in the dark in a water bath at 37°C according to the manufacturer’s instructions (Beyotime Institute of Biotechnology). The cells were then collected and underwent cell cycle analysis using a flow cytometer .LN229 cells  was used to estimate significant differences between groups. P<0.05 was considered to indicate a statistically significant difference. All experiments were performed a minimum of three times and data were analyzed using GraphPad Prism, version 5  and SPSS, version 13.0 .To investigate the tumorigenesis-associated molecular alterations in glioma, the microRNA expression levels were analyzed in 63 patients with grade II glioma and 135 patients with HGGs by microarray analyses. Among these microRNAs, miR-206 expression was observed to be downregulated as the degree of malignancy in gliomas increased , leading to the identification of cyclinD2. CyclinD2 was observed to share seven imperfect complementary sites with miR-206 and was identified to be important in the cell cycle; therefore, a luciferase reporter assay was designed to verify this. Subsequent to co-transfection with the miR-206 mimics and cyclinD2-3′UTR-plasmids, relative luciferase activity was observed to be significantly reduced (P<0.01; Based on the above analysis, the possible targets of miR-206 were searched with TargetScan ( P<0.01; . The luc P<0.01; . Finally P<0.01; . Based oAccording to the gene expression microarray, it was observed that with a higher degree of malignancy, cyclinD2 expression was significantly increased (P<0.0001; 0/G1 phase, whereas a significant reduction in cells in the G2 and S phases was observed compared with that in negative control cells (P<0.001, P<0.01 and P<0.01, respectively; 1/S transition in glioma cell lines via targeting cyclinD2.To investigate the biological function of miR-206 in the progression of glioma, a series of overexpression assays were conducted in the GBM cell line LN229. A colony formation assay indicated a significant reduction in colony formation in the miR-206-transfected cells compared with that in the negative control cells P<0.05; . In addiPrevious studies have indicated that microRNAs regulate gene expression and may also function as tumor suppressors or oncogenes . By bindIn the present study, miR-206 was observed to be downregulated in glioma based on microRNA microarray analysis. In addition, the overall survival of patients varied significantly depending on the expression levels of miR-206, suggesting that patients with a high expression of miR-206 had an improved prognosis, based on separate statistical analyses in grade II gliomas and HGGs. Therefore, it was hypothesized that miR-206 may be important in tumorigenesis and the progression of glioma.1 cyclins, including cyclinDs and cyclinEs, combined with cyclin-dependent kinases CDK4 and CDK6, have been reported to be activated in the late G1 phase and regulate G1/S transition (1 to S phase is commonly observed (0/G1 population and a reduction in the S phase population as compared with negative control cells. These results further demonstrated that miR-206 acted as a cell cycle inhibitor, as an increase in the levels of miR-206 expression significantly inhibited transition of LN229 cells from G0/G1 to S phase. In conclusion, cyclinD2 was a direct target of miR-206 and miR-206 regulated the cell cycle by promoting G1/S arrest and suppressing cell proliferation via targeting cylinD2 in gliomas.To further investigate the function of miR-206 in glioma progression, the target-predicting database Targetscan was searched and cyclinD2 was identified as a potential target of miR-206, which is associated with the cell cycle. miR-206 was previously reported to regulate cyclinD2 in rhabdomyosarcoma ,12, breaobserved . Based oIn conclusion, to the best of our knowledge, the present study was the first to demonstrate that miR-206 suppresses glioma formation and possibly targets the downstream complementary sites of cyclinD2 to inhibit cancer cell proliferation. In addition, the low expression of miR-206 in patients with glioma was demonstrated to be correlated with poor prognosis. Therefore, it was concluded that miR-206 acts as a tumor suppressor in glioma and regulates cell proliferation and cell cycle arrest by targeting cyclinD2. On the basis of observation and data analysis, miR-206 was suggested to be a novel candidate for use as a prognostic marker in patients with glioma and to have potential for use as a therapeutic target in gliomas.


Fasciola hepatica is considered an emergent human pathogen, causing liver fibrosis or cirrhosis, conditions that are known to be direct causes of cancer. Some parasites have been categorized by WHO as carcinogenic agents such as Opisthorchis viverrini, a relative of F. hepatica. Although these two parasites are from the same class (Trematoda), the role of F. hepatica in carcinogenesis is unclear. We hypothesized that F. hepatica might share some features with O. viverrini and to be responsible to induce proliferation of host cells. We analyzed the recently released genome of F. hepatica looking for a gene coding a granulin-like growth factor, a protein secreted by O. viverrini (Ov-GRN-1), which is a potent stimulator of proliferation of host cells. Using computational biology tools, we identified a granulin-like molecule in F. hepatica, here termed FhGLM, which has high sequence identity level to Ov-GRN-1 and human progranulin. We found evidence of an upstream promoter compatible with the expression of FhGLM. The FhGLM architecture showed to have five granulin domains, one of them, the domain 3, was homologue to Ov-GRN-1 and human GRNC. The structure of the FhGLM granulin domain 3 resulted to have the overall folding of its homologue the human GRNC. Our findings show the presence of a homologue of a potent modulator of cell growth in F. hepatica that might have, as other granulins, a proliferative action on host cells during fascioliasis. Future experimental assays to demonstrate the presence of FhGLM in F. hepatica are needed to confirm our hypothesis. F. hepatica or Fasciola gigantica, is a major public health problem in many tropical and subtropical regions. Recent reports have estimated that between 2 and 17 million people are infected, and 180 million people are at risk of infection; prevalence is particularly high in the Andean highlands of Peru, Ecuador, and Bolivia . As demonstrated in O. viverrini, the biliary damage predispose to develop cancer  that has been shown to be mitogenic at very low concentrations  has been associated with many aggressive cancers such as CCA and its overexpression is related to tumor growth, angiogenesis and resistance to apoptosis Demorrow . Of noteO. viverrini and F. hepatica, it would be possible that they may have similar biological products (i.e. growth factors). The discovery of growth-factor like molecules in F. hepatica may provide vital information and insights into the fundamental biology of this parasite, identify related pathways linked to fluke-host interactions and predict interactions from host factors into the disease. The aim of this study is to identify a potential growth-factor, topologically similar to both Ov-GRN-1 and PGRN, in F. hepatica genome by using computational biology tools.Given the close phylogenetic relationship between liver flukes such as F. hepatica GRN-like sequences we screened for closely related helminth parasite O. viverrini granulin (Ov-GRN-1) sequence in the F. hepatica genome available in the WormBase ParaSite website under the code PRJEB6687. The Blastp search returned 9 hits for the Ov-GRN-1 query. All of them were originated from the same scaffold numbered 891. The two top entries were selected because of their lower E-values cut-off resulting as of 1.2E−14 and 7.0E−11, respectively. Both entries corresponded to two different regions within the deduced protein of scaffold 891 whose transcript was identified as to BN1106_s891B000441.mRNA-1 (a product of the gene BN1106_s891B000441), which has 1959 bp and 9 exons. An upstream promoter was identified by Neural Network Promoter Prediction, which is compatible with the expression of this gene. The homologue protein of Ov-GRN-1 identified in the F. hepatica genome (translation ID BN1106_s891B000441.mRNA-1 in the WormBase ParaSite) has 652 residues, a molecular weight of 71.48 kDa and pI of 6.02  and the second on located from residues 161-223 (Domain 3). Domain 2 was 41 % identical to Ov-GRN-1 with a significant E-value 7.0E−11 while Domain 3 was 54 % identical to Ov-GRN-1 with a significant E-value 1.2E−14. Subsequent Blastp searching against the bovine genome showed that scaffold 891  displayed 28 % identity to bovine granulin isoform X2 (GenBank:XP_010814706.1), a predicted GRN protein. Therefore, FhGLM was identified as possibly encoding proteins containing the GRN conserved domain.To identify potential n Ov-GRN- sequenceOv-GRN-1, FhGLM was significantly longer (550 residues more than Ov-GRN-1) and it looks like a multi-homodomain GRN protein in contrast with Ov-GRN-1 that has a single GRN domain behind a secretory signal peptide  resulting in 54 % identity . The FhGLM-D3 has a composition of positively residues Arg and Lys (5.2 %) comparable to Ov-GRN-1 (7.8 %) but markedly different to human GRNC, which has no Arg and Lys residues in its composition. The content of Cys residues is similar in the Fh-GLM-D3 (22.6 %) compared to human GRNC (22.2 %), which is higher than content of cysteins in Ov-GRN-1 (12 %). The theoretical point isoelectric (pI) of Fh-GLM-D3 (5.76) and human GRNC (4.01) were notably lower than Ov-GRN-1 (8.29). In accordance to those findings, the theoretical charges at pH 7 showed that the Fh-GLM-D3 has an acidic profile (charge of −1.7) similar to the human GRNC (charge of −6) but opposed to Ov-GRN-1 (charge of 3.8).Other protein features were evaluated in the Ov-GRN-1 (102 residues) and Brugia malayi GRN (77 residues). The structural architecture in the two latter granulins are constituted by one GRN domain only. At the contrary FhGLM was predicted to have 5 GRN domains similarly to other organisms considered in our analysis and that present more than one GRN domain, including helminths Schistosoma japonicum and S. haematobium, Clonorchis sinensis, Ascaris suum, Haemonchus contortus, Caenorhabditis elegans, Strongyloides ratti, Echinococcus Granulosus and vertebrates including Mus musculus, Homo sapiens, Bos taurus.Comparative analysis with other GRN members showed that FhGLM is substantially longer than To examine sequence features of the candidate GRN family member, we performed a multiple sequence alignment of known GRN proteins from both mammalian hosts and helminths. Bovine and human PGRN were included as reference sequences for analysis.The alignment of Fh-GLM-D3 with other GRN family members showed that all GRN family members, and also Fh-GLM-D3, contained 12 conserved Cys residues arranged into four Cys pairs and flanked by two single Cys at both the amino and carboxyl termini  very closely with F. hepatica and the nematode H. contortus whereas the cestode E. granulosus was grouped closely with the blood flukes (Schistosoma) in a separate clade. The other subfamily grouped the trematodes O. viverrini and C. sinensis with the nematode S. ratti in a clade whereas GRNs from the nematodes A. suum, C. elegans and B. malayi were grouped in a separate clade.The evolutionary relationship between the Both the complete FhGLM (652 residues) and the Fh-GLM-D3 (52 residues) sequences were submitted to Phyre2 to build the corresponding structures. The complete FhGLM structure was constructed as follows: 58 % of residues were modelled at >90 % confidence using multiple-templates and applying both homology modeling and ab initio Ping 1.0 approach  as template, with 99.2 % confidence Fig. . The higThe local model quality was assessed by calculating the knowledge-based energy, which resulted quite favorable both in the Fh-GLM-D3 and the resolved human GRNC. In contrast, the knowledge-based energy of the complete FhGLM structure resulted in positive values suggesting some problematic or erroneous parts in the modeled structure. The quality findings mentioned above agreed with the assessment conducted using ProQ2 suggesting the good quality of the Fh-GLM-D3 structure and the limitations to obtain a good quality structure of the complete FhGLM  has been demonstrated to be a potent stimulator of cell proliferation and has been associated with cancer progression  was identified here is a homologue both of human GRN and Ov-GRN-1. In size and architecture terms the FhGLM, with 652 residues and 5 granulins, is similar to the human PGRN, which is composed of 543 residues and 7 GRNs. In contrast, the FhGLM is larger and has a more complex architecture than Ov-GRN-1, this latter is constituted of 102 residues and it contains only one GRN domain .Primary sequence analysis of Fh-GLM-D3 using SignalP suggested that such hypothetical protein lacked predicted N-terminal signal peptides for secretion via the classical ER/Golgi pathway , which might be specific and vital to establishing a parasitic life cycle. The phylogenetic relationship of this GRN stimulating cell growth factor supports a functional protein relationships rather than taxonomic relationships as previously suggested , constitutes a preliminary evidence that F. hepatica may have proliferative and mitogenic factors secreted to the tissue environment that might stimulate the cell proliferation, similar to O. viverrini and T. spiralis. As pro-granulins regulate cell proliferation, motility and inflammation; and they have an established role in the progression of ovarian and breast cancers  with topological features similar to its homologue in O. viverrini, suggesting that FhGLM might conserve its function as cell proliferative factor. Similarly, we found that the FhGLM was highly similar in topology and predicted biological features to the human progranulin, a factor that is related to tumorigenesis. In conclusion, our results suggest that F. hepatica genome contains a granulin gene that despite of lacking a signal peptide, it would code for a secretory protein. Its effect on proliferation of host cells either during the course of the disease or inducing directly a malignant process needs to be further investigated.We found by applying theoretical approaches a granulin-like molecule in the genome of O. viverrini (Ov-GRN-1) was retrieved from the UniProtKB (Swiss-Prot:ACJ83119.1). Genome analysis was conducted using the putative F. hepatica genome produced in the laboratory of Dr. Jane Hodgkinson at University of Liverpool . The accession number of the F. hepatica genome used in this work and deposited in the WormBase ParaSite was PRJEB6687. GRN like sequences were identified in the F. hepatica genome through a Blastp search of the draft genome contigs using protein sequence of Ov-GRN-1 from O. viverrini as query. Protein database search was activated and default terms were set for searching. An E-value cut off of 1 × 10−4 was used to define a significant hit. Promoter was searched by applying Neural Network Promoter Prediction  using GlyocoEP  .The structural architecture of GRN family members was obtained from entry IPR000118 at the Interpro 18.0 database  implemented in NCBI website. The amino acid sequences of GRN homologues in helminths identified by PSI Blast were retrieved from UniProtKB and GenBank. Similarly, the amino acid sequences of GRN family in mammal hosts were retrieved from UniProtKB for further analysis. The amino acid sequences of GRN homologues from helminths including F. hepatica candidate as well as GRN family from mammalian hosts were aligned using MUSCLE , individual GRN domains sharing the greatest identity with Fh-GLM-D3 were selected and aligned. Core granulin domains were compared and conservation of amino acids was analyzed using Seaview version 4 . For recognition of errors in the 3D-structure FhGLM constructed, ProSa was used  on Phyre2 (Kelley et al.


However, there is a paucity of information about how the MSC-MVs are up-taken by endothelial cells.Previous data have proven that microvesicles derived from hypoxia-induced mesenchymal stem cells (MSC-MVs) can be internalized into endothelial cells, enhancing their proliferation and vessel structure formation and promoting MVs were prepared from the supernatants of human bone marrow MSCs that had been exposed to a hypoxic and/or serum-deprivation condition. The incorporation of hypoxia-induced MSC-MVs into human umbilical cord endothelial cells (HUVECs) was observed by flow cytometry and confocal microscopy in the presence or absence of recombinant human Annexin-V (Anx-V) and antibodies against human CD29 and CD44. Further, small interfering RNA (siRNA) targeted at Anx-V and PSR was delivered into HUVECs, or HUVECs were treated with a monoclonal antibody against phosphatidylserine receptor (PSR) and the cellular internalization of MVs was re-assessed.The addition of exogenous Anx-V could inhibit the uptake of MVs isolated from hypoxia-induced stem cells by HUVECs in a dose- and time-dependent manner, while the anti-CD29 and CD44 antibodies had no effect on the internalization process. The suppression was neither observed in Anx-V siRNA-transfected HUVECs, however, addition of anti-PSR antibody and PSR siRNA-transfected HUVECs greatly blocked the incorporation of MVs isolated from hypoxia-induced stem cells into HUVECs.PS on the MVs isolated from hypoxia-induced stem cells is the critical molecule in the uptake by HUVECs. Cardiovascular and peripheral blood vessel diseases are the commonest conditions in the elderly. Usually, atherosclerosis is the underlying disease which is initiated and aggravated by the continuous defects of integrity in the vascular endothelium, resulting in the vessel occlusion and subsequent damage and dysfunction of the involved tissues and organs. Mesenchymal stem cells (MSCs) are adult stem cells characterized by their immuno-regulatory, hematopoiesis-supporting and angiogenesis-promoting activities. According to the reports, many tissues have been demonstrated to be isolated the MSCs, including bone marrow, adipose tissue, liver, muscle, amniotic fluid, placenta, umbilical cord blood, umbilical cord and dental pulp. In the Increasing clinical trials have been performed to testify the safety and effectiveness of MSCs in the management of these ischemic diseases –8. Howevin vitro proliferation and vessel structure formation and promoting in vivo angiogenesis as well [Interestingly, the transplanted MSCs will be exposed to the microenvirment of hypoxic and ischemic in these diseases. Previous study demonstrated that MSCs are able release large quantities of microvesicles (MVs) under a hypoxic and/or serum-deprivation condition . MVs fro as well . The ang as well  and a mo as well . MeanwhiRecent studies indicate the microvesicles was a vital mediator in the cell-to-cell communication, and internalization may be the key process. Accordingly, numerous researchers showed the contents of MVs vary greatly depending on the originate cells, nevertheless, all the MVs contain some endogenous substances including membrance traffic proteins , multivesicular bodies , intergins and tetraspanins . Additionally, the raft-lipids  and some signal transduction  also have been detected,15. KatrIn the present study, the potential pathway that hypoxia-induced MSC-MVs enter into human umbilical cord endothelial cells (HUVECs) has been probed and, the results here suggest that the interaction of phosphatidylserine (PS) on the MVs isolated from hypoxia-induced stem cells, with the PS receptor (PSR) on the HUVECs is largely responsible for the incorporation.In this study, all the Human Bone marrow samples and umbilical cords were collected after an informed consent was given, and in accordance with the Ethics Guidelines for Research Involving Human Subjects or Human Tissue from the General Hospital of Air Force. All procedures have been reviewed and approved by the Institutional Review Board (IRB) of Academy of Military Medical Sciences. All the participants provide their written informed consent to participate in the study. In the subject application stage, all the participants had learned the research content carefully, and provided the informed consents. And the related organization of Air Force General Hospital checked the informed consents, then the National Natural Science Foundation approved. The ethics committees/IRBs approve this consent procedure.2. The fresh medium was changed every 2–3 days, and the cells were sub-cultured at 90% confluence.The BMSCs were isolated and identified as previously described . BrieflyHUVECs were isolated and cultured according to the protocol of our lab . In brie2, 5%CO2, 94%N2) or normoxia for 72 hours and digested with 0.05% enzyme. Then the cells were washed twice with PBS, and trypan blue exclusion test was used to detect the cells viability. It found that the cells viability was >95%. Subsequently, the supernatants were collected and centrifuged at 1500×g for 15 minutes to remove the cell debris, followed by a filtration through a 0.22μm Super Membrane . The supernatants were then ultracentrifuged at 170,000×g (Beckman Coulter Optima L-100 XP Ultracentrifuge) for 5 hours at 4°C. The precipitate pellets were then washed twice in PBS and suspended in the apop buffer containing 5mM KCl, 1mM MgCl2, and 136mM NaCl. In selected experiments, isolated MVs were resuspended in 1ml PBS that mixed with 5μl DiI or 1μl CFSE , and incubated for 20 minutes in dark at room temperature. The labeling reaction was stopped by adding an equal volume PBS of 1% BSA. Then labeled microvesicles were washed twice with PBS and ultracentrifuged at 170,000×g for 5 hours at 4°C. The protein concentration of MVs was determined with the BCA protein assay kit  and accordingly, MVs were split into aliquots and stored at -80°C.MSC-MVs were harvested as previously reported with a mild modification . BrieflyThe identification of MSC-MVs was performed as previously described . MVs werTo further clarify the components of the vesicles, western blot was also performed. Firstly, The MVs and hypoxia-induced MSCs were respectively collected and lysed with 100μl RIPA buffer  supplement with 1μl protease inhibitor on ice for 30min. Then pyrolysis products of the cells were centrifuged at 15,000×g for 15min at 4°C and BCA protein assay kit was used to detect the protein concentration. 30μg protein was electrophoresed on 10% SDS-polyacrylamide gel, and transferred to a nitrocellulose membranes at 60 mA for 3 h. The membrane was subsequently washed and blocked with 5% skim milk, and incubated with diluted corresponding antibody  overnight at 4°C. Lastly, the HRP conjugated anti-rabbit IgG was used and exposed with an X-ray film.To observe the internalization process of MSC-MVs, HUVECs were seeded into glass-bottomed culture dishes specially designed for confocal observation in the presence of 10μg/ml DiI-labeled MVs and the culture was incubated for the indicated time-points. Unbound MVs were removed by extensive washing with PBS, followed by the cell fixation with 4% Paraformaldehyde for 15min at room temperature and pretreatment with 0.5% Triton-X100 for 10 minutes. The cells were then washed with 5% BSA in PBS with Tween 20 and incubated for 20min to block unspecific binding sites. A rabbit anti-human alpha Tublin antibody  at a dilution of 1:200 in PBS containing 0.5% BSA was added and the cells were incubated overnight at 4°C. After washing in PBS, a goat anti-rabbit IgG antibody conjugated FITC  was added and incubated for 60 minutes at room temperature. The nuclei were counter-stained with 1μmol/L 4',6-diamidino-2-phenylindole  for five minutes. The cells were observed with a Zeiss LSM 510 Confocal laser scanning microscope, and the images were analyzed by using the Zeiss LSM and volocity Demo software . The relative fluorescence intensity of DiI dye was assessed with a software Image-Pro Plus .Confocal microscopy was also used to detect PSR expression on HUVECs. Briefly, the cells were fixed and reacted with a rabbit monoclonal antibody against PSR  at a dilution of 1:200. The cells were washed in PBS and reacted with a FITC-conjugated secondary antibody. Then the cells were observed as described above.HUVECs were cultured in the presence of CFSE-labeled MSC-MVs that had been pretreated with mouse-originated monoclonal antibodies against human CD29 and CD44  at a dose of 1μg/ml and/or recombinant human Anx-V  at graded concentrations. The cells were collected by trypsin digestion and washed twice in PBS. At least 10,000 events were harvested with a flow cytometer . The data were analyzed with the WinMDI 2.9 software after the target events were gated according to the forward and side scatter corner signals. HUVEC co-cultured with unlabled MVs were served as control.4 HUVECs in a volume of 100μl were seeded into a 48-well plate, which was pre-coated with 100μl matrigel for 1 hour. The cells were then incubated at 37°C, 5% CO2 for 24 hours and tube formation was observed by microscopy. Further, to observe the involvement of PS in the incorporation of MVs into HUVECs, MVs (100μg/ml) were mixed with recombinant human Anx-V (10μg/ml)  and the reaction lasted for 1 hour at 37°C. Then the mixture was washed with apop buffer and then added into the culture and the tube formation was reassessed. Moreover, the human Anx-V mixed with HUVECs for 1 hour at 37°C, then the cells was washed twice and planted on 48-well plate that including martigel as the negative control. The number of the network structure was quantified by randomly selecting 5 field per well.Aliquots of 3×10Small interfering RNAs (siRNA) targeted at human Anx-V and the control siRNA were purchased from Santa Cruz Biotech (Cat No. sc-36324 & sc-37007). The siRNA targeted against human Anx-V and the non-silencing control siRNA were designed according to the sequences reported previously . TransfeAll values are expressed as the mean ± S.D. Statistical analysis was completed with student’s two-tailed unpaired t-test or one-way ANOVA using Graph pad Prism 5. A P-value of <0.05 was considered statistically significant.MVs derived from hypoxia-induced hBMSCs were collected by ultracentrifugation and identified by electron microscopy and flow cytometry. The results showed that the MSC-MVs were relatively homogenous in morphology, round- or cup-shaped with a diameter around 50–100nm . Flow cyAccording to above methods, the specific molecules were detected after MSCs had experienced serum-free and normoxic condition. Flow cytometry revealed that the MVs derived from the normoxia cells seemed not to react with Anx-V, suggesting no PS exposure on their surface .To quantify the proportion of MV internalization, CFSE- or Dil-labeled hypoxia-induced MSC-MVs (10μg/ml) were added to the culture of HUVECs that had reached to a confluence of around 80%, and the cells were then collected at different time points for flow cytometry and confocal observation. As shown in In fact, the CFSE positivity varied greatly when HUVECs from different donors were used. To standardize the experiments below, HUVECs from three subjects were selected. The observation time-point was set as 12 hours and the dose of MSC-MVs was used at 10μg/ml.The experiments above revealed that PS, CD29 and CD44 were present on the surface of the MSC-MVs . These mTo testify this assumption, CFSE-labeled hypoxia-induced MSC-MVs (10μg/ml) were pretreated with recombinant human Anx-V (10μg/ml), and/or antibodies against CD29 and CD44 (1μg/ml), followed by the addition into HUVEC culture. Twelve hours later, the cells were collected for flow cytometric analysis . The resTo further confirm the involvement of PS, graded concentrations of recombinant Anx-V were added together with CFSE-labeled MSC-MVs (10μg/ml) into the culture of HUVECs. Flow cytometric analysis showed that exogenous Anx-V inhibited the uptake of MSC-MVs by HUVECs in a dose-dependent manner . As showvs. 2.50±0.57, P<0.0001). Meanwhile, pre-treatment of MVs with Anx-V nearly totally abrogated their enhancing effect , p<0.0001; vs. siRNA-CTR (78.45±2.87)%, P<0.0001; n = 3). However, the down-regulation of Anx-V did not affect the incorporation of MSC-MVs into HUVECs  and the endosomal processing and emission of plasma membrane (exosomes) from activated and apoptotic cells . In the PS exposure is a prominent feature of MVs and this phenomenon has been discovered in MVs derived from platelets, red cells, multiple myeloma cells, melanocyts and other cell types , 36, 37.PS, the most well-studied marker of cells apoptosis, is translocated to the outer leaflet of plasma membrane, and can bind specially to a variety of surface receptors on the cells including phagocytes, epithelial cells, and endothelial cells. These receptors such as PSR, C. Elegan CED-1, the tyrosine kinase receptor MER, integrins and TIM family members, can recognize and link the PS directly or indrectly –45. Anx-PSR is an well-known classic receptor for PS. Furthermore, the tethering and ingestion of erythrocyte apoptotic bodies by vascular smooth muscle cells depend on the interaction of PS and PSR . AdditioIn conclusion, the present study provides definite evidences to support the view-point that PS on the surface of hypoxia-induced MSC-MVs and PSR on HUVECs are important participants in the internalization of hypoxia-induced MSC-derived MVs into HUVECs. Previous data have confirmed that the angiogenesis-promoting activities of MSCs are partially due to the interaction of the MSC-MVs with endothelial cells –13. The S1 FigThe events were collected with a flow cytometer and the single beads (green) and the doublets of beads (red) were gated for further analysis. The percentages of the positivity in contrast to an isotype antibody are indicated. X-axis: forward scatter corner signals showing the size of the gated events. Beads: Beads were collected for the determination of the gates. Beads+MVs: MVs conjugated with beads were collected for further determination of the gates for analysis. CTR: Beads reacted with Anx-V-FITC.(TIF)Click here for additional data file.S2 FigThe hollow diagrams represent the control and the solid ones represent the CFSE fluorescence intensities of the indicated cells. X-axis: the relative fluorescence of CFSE, and Y-axis: the number of events; CTR: Control, without MSC-MVs; MV: HUVEC+CFSE-MSC-MVs(10μg/ml).(TIF)Click here for additional data file.


In recent years, the bioactive factors were utilized in exercise and athletic skin injuries. In this research, the concentrated conditioned medium of hypoxia-preconditioned adipose mesenchymal stem cells, which is rich in bioactive factor, is applied in full-thickness skin defect model to evaluate the therapeutic efficacy. Adipose mesenchymal stem cells were harvested from the abdominal subcutaneous adipose tissues. The surface markers and the potential of differentiation were analyzed. The conditioned medium of hypoxia-preconditioned stem cells was collected and freeze-dried and then applied on the rat full-thickness skin defect model, and the healing time of each group was recorded. Haematoxylin and eosin staining of skin was assessed by microscope. The characteristics of adipose mesenchymal stem cells were similar to those of other mesenchymal stem cells. The concentration of protein in freeze-dried conditioned medium in 1 mL water was about 15 times higher than in the normal condition medium. In vivo, the concentrated hypoxia-preconditioned conditioned medium can reduce the wound size and accelerate the skin wound healing. The concentrated hypoxia-preconditioned adipose mesenchymal stem cell-conditioned medium has great effect on rat model of wound healing, and it would be an ideal agent for wound care in clinical application. It is well established that active life style is associated with improved quality of life. However, skin wound is one of the most common causes of inactivity (lack of movement) . The skiAdipose mesenchymal stem cells (AMSCs) have the ability to repair skin damage and promote wound healing. The requirements of cells culture limited the clinical application of stem cells. Furthermore, only a small percentage of cells will survive in damaged skin tissues. It is believed that the secretome of AMSCs plays an important role in skin wound healing , 3. The In this study, we performed experiments using concentrated hypoxia-preconditioned AMSCs-CM (Conc. Hypo-AMSCs-CM) to evaluate the effects of concentration and nonconcentration of Hypo-AMSCs-CM on the rat's full-thickness skin defect model.μm cell strainer. After centrifugation (400 g for 10 min), the cellular pellet was resuspended in Dulbecco's Modified Eagle Medium/10% fetal bovine serum placed in concentration 2–4 × 104 cells/cm2. The phenotype of AMSCs (passage 3) was assessed by indirect flow cytometry. The analysis  was performed using CD29, CD34, CD71, and CD90 as primary fluorescent antibodies and using IgG-FITC and IgG-PE as secondary antibodies. The negative control was cells without primary antibodies. To induce osteogenic and adipogenic differentiation, the medium was replaced with adipogenic or osteogenic differentiation medium, as described before (when AMSCs as passage 3 grew to approximately 90% confluence) [The abdominal subcutaneous adipose tissue was collected from the female Sprague-Dawley rats (250–300 g). The adipose tissue was minced and digested with collagenase  at 37°C for 40 min under constant shaking. The cellular suspensions were passed through a 100 fluence) . The dif2 generator, for 24 h at 37°C [As described previously, cells were cultured in the sealed box with an Anaeropack, a disposable oxygen-absorbed and COμm filter and then centrifuged at Amicon-Ultra-15 mL  at 4°C 4000 g for 30 minutes. The medium was concentrated about 5 times using ultrafiltration membrane of 3.5 kD with polyethylene glycol at 4°C. The conditioned medium was concentrated about 15 times as described above. Freezing-dried powder was prepared in the sterilized penicillin bottle using the Lyophilizer . The concentration of protein in conditioned medium was measured by NanoDrop 2000 .The AMSCs culture dishes were washed three times with PBS and cultured overnight in culture medium consisting of DMEM/F-12, 0.4% rat serum. After 24 hours of incubation, the cultured medium was collected and then the fresh low-serum medium was added. The cultured medium of AMSCs was filtered using a 0.22 All animal experiments were conducted with the approval of Nanjing Medical University Ethics Committee and were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . The full-thickness skin defect model used in this study is identical to the model previously described [t-test analysis was applied for measuring the data, and the regression analysis was used for the relationship between the time and the percentage of wound healing. P < 0.05 was considered to be statistically significant.The results are expressed as mean ± standard deviation (SD). Statistical analysis was performed using analysis of variance (ANOVA) test by SPSS17.0, as appropriate. The 6/mL from abdominal fat. Primary AMSCs were cultured for 7 days  and between Hypo-AMSCs-CM and control groups (P < 0.001). Percentage of wound healing was evaluated by measurement of “healing area/initial wound area × 100%” on 7, 14, and 21 days after treatment in three groups ; that in Hypo-AMSCs-CM group was y = 0.0444x + 0.1108 ; and that in the control group was y = 0.0442x + 0.0688 . There were no differences between the Conc. Hypo-AMSCs-CM and Hypo-AMSCs-CM groups (P = 0.299). The Conc. Hypo-AMSCs-CM treatment improved wound healing when compared with that of the Hypo-AMSCs-CM group at 7 (P < 0.001) and 14 (P < 0.001) days , 14 (P < 0.001), and 21 days (P = 0.0005) . We first freeze-dried the conditioned medium of AMSCs, so the multiple concentration of conditioned medium could be made for clinical application.The applications of culture medium of other cells have also been widely used in wound healing. We searched the MEDLINE and EMBASE databases, using the “conditioned medium” and “skin wound” as search terms. All eligible studies were parallel-controlled trials about conditioned medium of stem cell in skin wound healing (see Supp. l in Supplementary Material available online at However, the umbilical cord Wharton's jelly cells  and boneAMSCs were located in perivascular niches, close to vascular structure in fat. In vivo, the oxygen concentration of fat tissue is 2%–8% . The lowSkin is the first line of body's defense in the protection of foreign invasion, as the skin is exposed to pathogens in the macroenvironment. The unexpected skin injury may affect circulative exercise training, thus disrupting normal training rhythm. Serious injury could potentially end athlete's whole sports career. According to the skin wound severity, the healing takes around 2 weeks generally. Many treatments have been proven to accelerate the healing, such as tissue engineering , stem ceThe Hypo-AMSCs-CM could be powdered through the concentrated and lyophilized process, and the high concentrated Hypo-AMSCs-CM, made by the powder, may be more effective in the skin wound treatment of the rat model.Supplementary Table 1: The summary of application of culture medium in wound healing. The sources of culture medium is most from adipose-derived stem cells, bone marrow mesenchymal stem cells and Wharton's jelly mesenchymal stem cells. And the hypoxia is the most common preconditioned way in cell culture. The growth factor in culture medium promote wound healing more rapid compare with placebo and stem cells, and hypoxia preconditioned could enhance the wound healing function of culture medium.